Identification of acetylated diether lipids in halophilic Archaea

Abstract As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea‐type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn‐glycerol 1‐phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O‐acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first‐time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects.

Database searches have indicated that YvoF homologs exist in many Halobacteria as well (Linde et al., 2016), which was quite surprising as acetylated ether lipids have not been known to exist in Archaea so far. Halobacterial YvoF sequences share an overall identity of about 50% among each other, and about 35%-45% with bacterial YvoF representatives; however, at the acetyl-CoA binding site, the similarity is significantly higher ( Figure A1). Since confirmation of the activity of YvoF from Halobacteria as well as identification of acetylated ether lipids within this phylogenetic clade has still been missing, we purified YvoF from Haloferax volcanii YvoF (hvYvoF) and verified acetyltransferase activity with bacterial C20 monoether lipids as substrate. We subsequently performed mass spectrometry (MS) to tackle the identification of acetylated ether lipids in extracts from H. volcanii and Halobacterium salinarum. Our results let us postulate a pathway for the synthesis of acetylated diether lipids in Halobacteria ( Figure 1b). We also discovered further acetylated phospholipids in the two halobacterial strains and discuss their occurrence in the context of YvoF.

| Cloning
hvYvoF was amplified by a polymerase chain reaction from H. volcanii H1424 genomic DNA. The primers used for amplification and cloning are given in Table A1. The produced fragments were cloned via NdeI/ XhoI into the pTA1228 expression vector (Allers et al., 2010) providing a C-terminal hexahistidine (His) 6 tag.

| Transformation procedures
Transformation of Escherichia coli for cloning was performed using a standard protocol for chemically competent cells (Sambrook et al., 1989). Production of competent cells of H. volcanii and polyethylenemediated transformation was performed as described by Cline et al. (1989). The used solutions and media were produced accordingly.

| Production and purification of recombinant hvYvoF
Heterologous gene expression was performed in H. volcanii H1424 (Allers et al., 2010). To this end, 81.7 mg L-tryptophan were dissolved in 360 mL hv-YPC medium (1.11 mM final concentration) by shaking at 180 rpm and 42°C in 3 L flasks. The medium was inoculated with an overnight culture (40 ml from the preculture diluted to 1 OD 600 ) of transformed H. volcanii cells to 0.1 OD 600 and shaking was continued until 0.5 OD 600 was reached. Gene expression was induced a second time by adding 2 mM L-tryptophan (final concentration) in 18% saltwater and incubation was continued for 16 h. Cells were harvested by centrifugation, resuspended in 40 ml 50 mM Tris, pH 8, 600 mM NaCl, 10 mM imidazole, and disrupted by sonication until the suspension was nonturbid anymore. Cell debris was removed by centrifugation. The His-tagged protein was purified from the clarified cell extract by immobilized metal ion affinity chromatography (IMAC).
An ÄKTApurifier system with a HisTrap FF Crude column (5 ml; Cytiva) was used, and a linear gradient of imidazole (10-500 mM) in 50 mM Tris, pH 8, 600 mM NaCl was applied to elute the protein. To remove interfering salts and imidazole, the protein was subjected to dialysis against 50 mM Tris, pH 8.0, and 600 mM NaCl. Protein concentrations were determined by absorbance spectroscopy using a Jasco V650 spectrophotometer. The molar extinction coefficients ε 280 and the molecular weight were calculated from the amino acid sequence using ProtParam (Gasteiger et al., 2005). Purified protein was dropped into liquid nitrogen and stored at −80°C. UV-Vis spectra for analysis of bound ligands were recorded using 10 µM protein (final concentration) in 50 mM Tris, pH 8, 600 mM NaCl (200-600 nm; response time 0.96 s; scan rate 40 nm min −1 ; bandwidth 2 nm).
2.5 | Nano differential scanning fluorimetry (nanoDSF) 2.6 | 14 C-GGG activity assay 14 C-GGGP was synthesized as described previously (Linde et al., 2016) in 50 mM Tris, pH 8.0, 10 mM MgCl 2 , 0.2% Tween80, and dephosphorylated to 14 C-GGG by adding 1 U of calf intestinal phosphatase (CIP) and further incubation at 40°C for 1 h. To test the activity of the purified hvYvoF enzyme, 1 µM hvYvoF, 0.25 mM acetyl-CoA (Sigma), and varying concentrations of NaCl were added to the synthesized 14 C-GGG (2.5 µM; 37.5 nCi) and incubated at 40°C. To visualize time-dependent activity, the reaction was stopped at different time points by the addition of chloroform. The products were extracted according to the method of Bligh and Dyer (Bligh & Dyer, 1959) as modified by Kates (1986

| RP-UHPLC-ESI-MS
The TLE was dissolved in 2 ml of DCM:MeOH (5:1, v:v) and 400 µl (20% of the TLE) was transferred into a new vial. C46-GTGT standard (Huguet et al., 2006)  ( Figure A2). The purified protein solution was colored pink, indicating that carotenoids such as lycopene and bacterioruberin, which are abundant in H. volcanii cells (Ronnekleiv, 1995), were copurified with hvYvoF. We assume that due to their isoprenoid nature, carotenoids bind to hvYvoF in the binding pocket of its isoprenoid substrate. Following this assumption, the UV-Vis spectrum of purified hvYvoF in the range of 450-550 nm is characteristic of bacterioruberin ( Figure A3) (Dummer et al., 2011). In thermal denaturation experiments using differential scanning fluorimetry (nanoDSF), hvYvoF showed cooperative transition curves ( Figure A4A). As expected for a protein from a halophilic organism, hvYvoF was significantly stabilized by increasing salt concentrations. The apparent midpoint temperature of the unfolding transition raised by about 27 K when NaCl was increased from 0 to 2000 mM ( Figure A4B).
To verify acetyltransferase activity, we performed an activity assay with a 14 C-labeled substrate as described previously (Linde et al., 2016). In brief, we synthesized radiolabeled geranylgeranylglyceryl phosphate ( 14 C-GGGP), which is the Archaea-type ether lipid as identified in B. subtilis, but with a C20 instead of a C35 isoprenoid. 14 C-GGGP was incubated with CIP, generating geranylgeranylglycerol ( 14 C-GGG), and hvYvoF as well as acetyl-CoA as donors for the acetyl group. The reaction was stopped by chloroform addition at different points in time. Afterward, the products were extracted, separated by thin-layer chromatography (TLC), and visualized with a phosphoimager system (Figure 2).
TLC analysis showed that GGG was gradually diacetylated (to Ac 2 -GGG) by hvYvoF with increasing incubation time. The monoacetylated intermediate Ac-GGG was scarcely detected. Diacetylation such as previously observed in B. subtilis (Figure 1a) is possible in this case, as two hydroxyl groups are available for acetylation when using GGG as substrate. In Archaea, however, the second hydroxyl group is normally occupied by another isoprenoid chain, which is also seen later in MS experiments with in vivo samples. Because hvYvoF is a protein from a halophilic organism, we tested the dependence of activity upon increasing salt concentration in the assay ( Figure A5).
As expected, the activity was significantly increased in the presence of salt, which became most clearly visible in the faster decrease of the substrate GGG over the incubation time.

| Identification of acetylated diether lipids in Halobacteria
To identify what kinds of acetylated ether lipids occur in vivo in  Figure 3 exemplarily shows the UHPLC elution profile and MS analysis for the H. volcanii extract, the data for H. salinarum are shown in Figure A6.
The results are summarized in Table 1.
The mass spectral fragmentation patterns indicate that besides regular archaeol (AR, corresponding to saturated DGGG or 2,3diphytanyl-sn-glycerol; cf.  would prove that it is YvoF that produces the Ac-AR and Ac-AG we found in vivo, but because of the solubility problems and because AR and AG are difficult to obtain, we refrained from such experiments. Nevertheless, together with previous data (Linde et al., 2016), our novel results strongly suggest that YvoF can acetylate a large number of glycerol derivatives, most likely also AR and AG. Previous results indicate that YvoF is a membrane-associated protein (Linde et al., 2016), making it likely that lipids are its primary substrate.
Another direct evidence would be the creation of a yvoF knockout strain in a halobacterial species, which is a laborious task, with no certainty that such a knockout strain would be viable and creatable at all. As an alternative to this, we have analyzed the content of Ac-AR and Ac-AG in an H. volcanii strain that overexpresses yvoF, namely the strain we used for the production of the hvYvoF protein, following the hypothesis that this strain might have elevated levels of acetylated lipids. However, MS analysis of TLEs from this strain revealed no significant difference in weight. One reason for this observation might be that acetylation is regulated by additional means other than the expression level of YvoF.

| CONCLUSION
Our results demonstrate that halobacterial YvoF exhibits GGG acetyltransferase activity in vitro, just like its homolog from B. subtilis (Linde et al., 2016). This matches our mass spectrometric identification of Ac-AR, Ac-AG, and various unsaturated derivatives thereof in H. volcanii and H. salinarum. We suggest that YvoF is responsible for acetylating these lipids in both species (the sequence identity of their YvoF protein sequences is 67%), and that acetylated ether lipids might also occur in many other Halobacteria that possess a YvoF homolog.
As outlined in our proposed pathway (Figure 1b), our findings suggest that YvoF catalyzes acetylation of diether lipids in Halobacteria after the second isoprenoid chain has been added by DGGGPS because we were unable to detect any acetylated monoether lipids. DGGG might be much preferred over GGG by YvoF in vivo. Experiments with B. subtilis YvoF indicated that it is a membrane-associated protein (Linde et al., 2016), which makes it likely that it acts on lipids that are located there. Most likely, acetylation happens in parallel to the reduction of the isoprenoid moieties by DGGGPR and the attachment of glycerol as a polar head group, as we found a series of different partially unsaturated Ac-AR and Ac-AG derivatives. We suppose that the acetyl group might serve alone as a small headgroup that reduces the polarity of the glycerol core of AR or serves to modify the polarity of the glycerol headgroup in the case of AG. It remains elusive how acetylation is in balance with the attachment of other head groups which cannot be acetylated.
According to a previous study (Sprott et al., 2003), the main polar membrane lipids of H. volcanii are archaetidylglycerol methylphosphate (AG-PCH 3 ; 44% of total lipid) and AG (35%), followed by sulfated glycolipids (14%), archaeal cardiolipin (5%), and archaetidic acid (2%). As a rough estimation for H. volcanii, the content of fully T A B L E 1 Identified ether lipids in Halobacteria 1 Compounds were found with up to eight unsaturations, which is indicated by dashed double bonds. 2 The theoretical (theo.) mass is calculated for the NH 4 + adducts and is given for the saturated molecules only.
saturated Ac-AR was about 10% of that of the detected amount of AR, and the amount of Ac-AG was 40% of that of AG. Based on these assumptions, the total content of acetylated ether lipids in a low percentage range seems reasonable and might be similar in H.

ACKNOWLEDGMENTS
We thank 2bind GmbH for access to the Prometheus NT.48 instrument (NanoTemper Technologies). We also thank Christiane Endres, Sonja Fuchs, Sabine Laberer, and Jeannette Ueckert for technical assistance. We are grateful to Reinhard Sterner for the critical reading of the manuscript and to Sébastien Ferreira-Cerca for providing the halobacterial strains and the expression vector. Open Access funding enabled and organized by Projekt DEAL.

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All the data are available in this published article.

ETHICS STATEMENT
None required.    (2) 14 C-GGG, no acetyl-CoA, no enzyme (GGG control); (3) 14 C-GGG, unlabeled acetyl-CoA (positive control for acetylation); (4) unlabeled GGG, 14 C-acetyl-CoA; (5) monoacylglycerol, 14 C-acetyl-CoA. For detailed reaction conditions, see Section 2. Note that for technical reasons, samples 2 and 3 contained 300 nCi of radioactivity, all other samples 40 nCi. To obtain spots of equal intensity, the sample volume applied to the TLC plate was reduced for samples 2 and 3. In each lane, 30 nCi were applied to the plate. The identities of the spots are labeled. (Ac-GGG), the position of Ac-GGG, which was only produced in minor amounts in lanes 3 and 4 and is therefore not visible; ?, putative identification, the identity of the spot has not been confirmed within this study or previously by MS; GGG, geranylgeranylglycerol; MAG, monoacylglycerol; hvYvoF, Haloferax volcanii YvoF; TLC, thin-layer chromatography.

ORCID
F I G U R E A11 Detection of saturated and unsaturated archaetidylglycerols (AGs). Extracted ion chromatograms (0.1 Da width) of AG analogs with 1-8 unsaturations (panel i-ix; uns, unsaturated; numbers in parentheses denote the number of carbons in both isoprenoid chains and the number of unsaturations in both chains) in Haloferax volcanii (a) and Halobacterium salinarum (b). Black arrows point to the respective detected chromatographic peaks, whereas red arrows show the expected retention time for compounds that could not be detected. Minor peaks at earlier retention times represent isotopologues with two 13 C atoms in acetylated unsAR with one more unsaturation or other compounds within the extracted mass window.